The long term objectives of this proposal are to define protein-protein interactions and signaling cascades that are likely to regulate globin gene expression. To identify these novel interactions and cascades, the zebrafish has been developed as a genetic model system to study globin gene expression. Five complementation groups of zebrafish mutants have been obtained with specific globin expression defects. The cloning of these mutant genes in a comparative approach for studying the mechanism of globin genes and all vertebrates should provide an in-depth understanding of how normal globin gene expression is accomplished in vivo. This proposal has two specific aims: 1) to characterize the phenotype of the five complementation groups of zebrafish mutants with defective globin expression. This will involve the evaluation of mutants for globin chain imbalance and defects in globin switching; an examination of the mutants for defects in the expression of other known genes that regulate globin expression; and the creation and examination of null alleles of each complementation group. 2) To isolate and characterize the defective genes for each complementation group. This will involve obtaining genetic markers that are within one cM of each affected gene; to utilize candidate and positional cloning techniques to isolate the mutant genes, making these as human-zebrafish-pufferfish regions of synteny; and to identify gene loci that interact with these mutant genes by performing screens for dominant suppressors.